Small RNAs in the peripheral blood discriminate metastasized from non-metastasized seminoma.

ABSTRACT

BACKGROUND: We aimed to better discriminate metastasized (lymphogen/occult/both combined) from non-metastasized seminoma based on post-transcriptional changes examined in the peripheral blood. METHODS: Total RNAs including small RNAs were isolated from the peripheral blood of patients suffering from metastasized testicular tumours (lymphogen, n = 5, clinical stage IIb/c; occult, n = 5, clinical stage I) and non-metastasized patients (n = 5, clinical stage I). Small RNA next generation sequencing (SOLID, Life Technologies) was employed to examine post-transcriptional changes. We searched for small RNAs showing at least 50 reads and a significant ≥ 2-fold difference using peripheral blood small RNAs of non-metastasized tumours as the reference group. Candidate small RNAs were examined in univariate logistic regression analysis and combinations of two small RNAs were further examined using support vector machines. RESULTS: On average 1.3 x 10(7), 1.2 x 10(7) and 1.2 x 10(7) small RNA reads were detectable in non-metastasized, lymphogen and occult metastasized seminoma, respectively of which 73-76% remained after trimming. From these between 80-82% represented annotated reads and 7.2-7.8% (1.6-1.7 x 10(4)) were annotated small RNA tags. Of them 137 small RNAs showed > 50 reads and a ≥ two-fold difference to the reference. In univariate analysis we detected 33-35 different small RNAs which significantly discriminated lymphogen/occult/combined metastasized from non-metastasized seminoma and among these different comparisons it were the same small RNAs in 44-79%. Many combinations of two of these small RNAs completely discriminated metastasized from non-metastasized seminoma irrespective of the metastasis subtype. CONCLUSIONS: Metastasized (either lymphogen or occult) seminoma can be completely discriminated from non-metastasized seminoma with a combination of two small RNAs measured in the peripheral blood.